Transcripts for two tetrahydrobiopterin (BH4) synthetic enzymes, GTP cyclohydrolase 1 (GCH1) and sepiapterin reductase, and a BH4 recycling enzyme quinoid dihydroypteridine reductase, are upregulated in dorsal root (DRG) neurons after peripheral nerve injury. This induction is associated with an increase in BH4 in the DRG. BH4 is an essential cofactor for the aromatic amine hydroxylases that produce 5- hyrdoxytryptamine, norepinephrine and dopamine, and for all nitric oxide synthases. The nerve injury-induced increase in BH4 is prevented by systemic administration of a GCH1 inhibitor, diamino hydroxypyrimidine (DAHP). DAHP has no analgesic effects in naove animals but produces a marked reduction in pain-related behavior in rodents with peripheral nerve lesions and inflammation. Intrathecal BH4 itself produces acute pain hypersensitivity. Using an analysis of single nucleotide polymorphisms we have also identified a haplotype in human GCH1 that is pain protective in patients after surgery for chronic back pain and associated with reduced pain sensitivity in healthy subjects. Based on these data we hypothesize that BH4 contributes to the initiation and maintenance of neuropathic and inflammatory pain. The aim of this proposal is to: 1. Study where and when BH4 induction occurs in the DRG in pain-related rodent models and what kinds of stimuli are responsible, 2. Characterize the behavioral consequences of deletion, inhibition or overexpression of GCH1 selectively in adult primary sensory neurons, and 3. Identify the mechanisms in sensory neurons by which BH4 produces pain. We will study the time course and cellular localization of changes in the expression and activity of BH4 synthetic and recycling enzymes in the DRG in response to tissue inflammation and partial peripheral nerve injury (Aim 1). To elucidate the specific role of BH4 in sensory neurons we will employ mice that selectively overexpress, or have a deletion of GCH1 in adult primary afferents, as well as mice that overexpress the endogenous inhibitor of GCH1, GFRP, using tamoxifen-inducible DRG neuron-specific Cre-recombinase technology (Aim 2). nNOS is upregulated in association with the increase in BH4 and we will test if BH4 produces pain by producing an increase in NO and calcium influx. We will use primary cultures of DRG neurons to explore the direct action of BH4 on these neurons and the downstream effectors responsible. The proposal is designed to explore the molecular mechanisms responsible for pain and identify novel targets for the development of new analgesics. Relevance: Persistent pain is an enormous problem due both to the suffering experienced by patients and the high economic cost to society. Because current therapy is often ineffective or associated with adverse side effects, more efficacious analgesics are required. This grant aims both to understand the mechanisms responsible for pain, and validate a particular enzyme, GCH1, as a target for developing novel analgesics.